Chimeric promoters capable of mediating gene expression in plants upon pathogen infection and uses thereof

ABSTRACT

Described are synthetic promoters capable of mediating gene expression in plants upon pathogen infection. Furthermore, recombinant genes and vectors comprising said chimeric promoters as well as host cells transformed with such chimeric promoters, recombinant genes, or vectors are provided. Additionally, diagnostic compositions and kits comprising such chimeric promoters, recombinant genes, vectors or cells are described. Provided are further methods for the identification of compounds being capable of activating or inhibiting genes that are specifically expressed in plants upon pathogen infection employing the above described means. Furthermore, transgenic plant cells, plant tissue, and plants containing the above-described chimeric promoters, recombinant genes, and vectors as well as the use of the aforementioned chimeric promoters, recombinant genes, vectors and/or compounds identified by the method of the invention in plant cell and tissue culture, plant breeding, and/or agriculture are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of patent application Ser. No.13/225,509 filed Sep. 15, 2011, and issued as U.S. Pat. No. 8,580,943Nov. 12, 2013, entitled CHIMERIC PROMOTERS CAPABLE OF MEDIATING GENEEXPRESSION IN PLANTS UPON PATHOGEN INFECTION AND USES THEREOF, which isa continuation-in-part-of patent application Ser. No. 09/831,272, filedAug. 13, 2001, entitled CHIMERIC PROMOTERS CAPABLE OF MEDIATING GENEEXPRESSION IN PLANTS UPON PATHOGEN INFECTION AND USES THEREOF, andissued as U.S. Pat. No. 8,013,138 on Sep. 6, 2011, which is a NationalStage Entry of PCT/EP99/08710, filed Nov. 12, 1999, entitled CHIMERICPROMOTERS CAPABLE OF MEDIATING GENE EXPRESSION IN PLANTS UPON PATHOGENINFECTION AND USES THEREOF, both of which are incorporated herein intheir entirety.

FIELD OF THE INVENTION

The present invention relates to synthetic promoters capable ofmediating gene expression in plants upon pathogen infection. The presentinvention also relates to recombinant genes and vectors comprising saidchimeric promoters as well as to host cells transformed with suchchimeric promoters, recombinant genes or vectors. The present inventionadditionally relates to diagnostic compositions and kits comprising suchchimeric promoters, recombinant genes, vectors or cells.

The present invention also relates to methods for the identification ofcompounds being capable of activating or inhibiting genes that arespecifically expressed in plants upon pathogen infection employing theabove described means. Furthermore, the present invention relates totransgenic plant cells, plant tissue and plants containing theabove-described chimeric promoters, recombinant genes and vectors aswell as to the use of the aforementioned chimeric promoters, recombinantgenes, vectors and/or compounds identified by the method of theinvention in plant cell and tissue culture, plant breeding and/oragriculture.

BACKGROUND OF THE INVENTION

The engineering of disease resistance in crops is a major focus of plantbiotechnology. One of the most promising approaches to this problem isto engineer defense reactions that are closely related to naturaldefense mechanisms such as hypersensitive cell death at infection sites,where the cells immediately surrounding an infection site die in orderto prevent further spread of the pathogen (Strittmatter, Bio/Technology13 (1995), 1085-1089). The controlled generation of highly localizednecrotic lesions depends, however, on restricting any cytotoxic activityto the infection sites. This therefore requires promoters that arerapidly and locally responsive to pathogen attack but that also shownegligible activity in uninfected tissues.

Initial attempts using large promoter fragments frompathogenesis-related genes such as prp1-1 have suffered from thedisadvantage that it is difficult to isolate a promoter that is totallypathogen specific with substantially no activity in non-infected tissue(Strittmatter, 1995). It seems likely therefore that very few, if any,naturally occurring promoters will be suitable for this purpose.

Recent advances in the detailed study of defense related genes haveidentified a number of functionally defined cis-acting regulatory DNAelements within pathogen inducible promoters (Korfhage, The Plant Cell 6(1994), 695-708, Raventos, Plant J. 7 (1995), 147-155, Rushton, EMBO J.15 (1996), 5690-5700). A number of cis-acting elements that arenecessary for the response to pathogens have been defined. These includeBoxes P and L from the parsley PAL genes (Logemann, Proc. Natl. Acad.Sci. USA 92 (1995), 5905-5909), Boxes H and G from soybean PAL and 4CL(Loake, Proc. Natl. Acad. Sci. USA 89 (1992), 9230-9234), together witha number of less well defined elements. However, while it was shown fora number of such cis-acting elements that they are necessary forelicitor inducibility it was not known whether these elements aresufficient to direct pathogen-induced expression in plant cells andplants on their own. Recently, it has only been shown for the Box W1from parsley (Rushton, EMBO J. 15 (1996), 5690-5700) and ERE from themaize Prms (Raventos, Plant J. 7 (1995), 147-155) that four copies ofthese elements alone are sufficient to direct elicitor responsiveexpression to some extent in transient gene expression assays. However,inducibility and background level of expression of the constructsinvestigated in Rushton, 1996 and Raventos, 1995 greatly varied and atbest an about 10-fold induction of reporter gene expression was observedthat may not be sufficient to supply the above-describedbiotechnological needs. Accordingly, it was unclear whether these or anyother cis-acting elements may be useful to specifically suppress orconfer local gene expression in plants upon pathogen infection.

Thus, the technical problem of the present invention is to providepromoters that are rapidly and locally responsive to pathogen attack butshow negligible activity in uninfected parts of the plant and that canbe used for engineering of disease resistant crops.

The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

SUMMARY OF THE INVENTION

Accordingly, the invention relates to a chimeric promoter capable ofmediating local gene expression in plants upon pathogen infectioncomprising

(i) at least one cis-acting element sufficient to directelicitor-specific expression comprising the nucleotide sequence of anyone of SEQ ID NOS: 3 to 16, and

(ii) a minimal promoter.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the present invention, and theattendant advantages and features thereof, will be more readilyunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying drawings wherein:

FIG. 1 shows a restriction map of the plasmid ms23 (Sprenger, 1997) (SEQID NO: 17);

FIG. 2 shows an overview cartoon of the plasmid ms23. The Gus reportergene and minimal −46 CaMV 35S promoter are shown, as are restrictionsites found in the polylinker sequence situated 5′ to the minimalpromoter. The distances (in base pairs) between the restriction sitesare also shown;

FIG. 3 shows an overview cartoon of the plasmid pGPTV. The Gus reportergene and minimal −46 CaMV 35S promoter are shown as are the SpeI andXbaI sites used in making the constructs employed. The nptII selectionmarker is also indicated, as are the left and right T-DNA borders (L andR). The terminators (pApnos and pnos) and promoter driving the nptIIgene (pAg7) are also shown;

FIG. 4 shows elicitor inducibility of chimeric promoters containing BoxE17 and derivatives thereof. GTAC motifs in forward and reverseorientation are underlined. Deleted bases are depicted as Ø. Thedepicted fragments are located 12 bp upstream of the 35S minimalpromoter. The monomers of the dimeric construct A109 are separated by a6 bp restriction site (SEQ ID NOS:27-31);

FIG. 5 shows elicitor inducibility of chimeric promoters containingdissected Box E17 elements. Starting from a Box E17 containing chimericpromoter (H149), chimeric promoters were constructed having 6nucleotides deleted from the 5′-end of Box E17 (B175), 7 nucleotidesfrom its 3′-end (C175) or comprising both deletions (C175).Additionally, a promoter was tested comprising a 445 bp Eli17 promoterfragment from which the 27 bp Box Eli17 element was deleted (A175).Relative and absolute elicitor induction values are given that weremeasured in transient expression assays (SEQ ID NOS:32-35);

FIG. 6 shows a cut-out of the polylinker of the vector ms23. Formeasuring the influence of the distance to the 35S minimal promoter BoxE17 or its dimer was inserted into eight different restriction sites(SEQ ID NO: 36);

FIGS. 7a and 7b show elicitor inducibility of Box E17 depending on thedistance to the 35S minimal promoter, as illustrated in FIG. 6. FIG. 7ashows the induction upon elicitor treatment for the BamHI, ClaI, EcoRI,XbaI, and SalI constructs. FIG. 7b shows, in another experiment, theinduction for elicitor treatment for the SapI-dimer, HindIII-dimer,BamHI, ClaI, EcoRI, SpeI, XbaI, and SalI constructs. ms23, in FIGS. 7aand 7b , represents the vector only containing the minimal promoter asnegative control; and

FIG. 8 shows expression characteristics of transgenic plants transformedwith reporter gene constructs comprising chimeric promoters withtetramers of some cis-elements of the present invention. For comparisonthe GCC-Box element is included (see Example 1). The backgroundexpression levels are quantified as being low (barely detectablebackground expression), medium (visible background expression butinduction by pathogens is clearly visible over the background) or veryhigh (extremely high expression such that induction by pathogens isdifficult to detect). A minus indicates no detectable expression, a plusindicates inducible expression and “nt” not tested.

DETAILED DESCRIPTION OF THE INVENTION

The term “capable of mediating local gene expression in plants uponpathogen infection” as used herein means that said promoter is capableof controlling the expression of a heterologous DNA sequence atinfection sites, analogous or closely related to the controlledexpression of pathogen related genes which are involved in the naturalresistance in most incompatible host/pathogen interactions, such as thehypersensitive cell death at infection sites of a part of a plant. Thus,the chimeric promoter of the invention is characterized by itscapability of mediating localized transcriptional activation selectivelyin response to pathogen attack or in response to stimuli that mimicpathogen attack such as elicitors prepared from, e.g., pathogens such asfungi or bacteria or derivatives thereof. The transcriptional activationby the chimeric promoter of the invention may also occur in cellssurrounding the actual infection site due to cell-cell interactions. Thechimeric promoter of the invention may advantageously not or only to asmall extent be inducible upon other stimuli such as abiotic stress.Preferably, the induction from the chimeric promoter upon pathogenattack or elicitor treatment is at least about 10-fold higher,preferably 20-fold higher and particularly 30-fold higher than itsactivation, if any, by abiotic stress.

However, the expression specificity conferred by the chimeric promotersof the invention may not be limited to local gene expression due topathogens, for example, they may be combined with further regulatorysequences that provide for tissue specific gene expression. Theparticular expression pattern may also depend on the plant/vector systememployed. However, expression of heterologous DNA sequences driven bythe chimeric promoters of the invention predominantly occurs uponpathogen infection or treatment with a corresponding elicitor unlesscertain elements of the invention were taken and designed by the personskilled in the art to control the expression of a heterologous DNAsequence in certain cell types.

The term “cis-acting element sufficient to direct elicitor-specificexpression” denotes a short stretch of a DNA preferably between 6 and 35nucleotides in length that when combined with a minimal promoter such asthe CaMV 35S minimal promoter (positions −46 to +8) is capable ofdirecting high level elicitor-specific expression of a heterologous DNAsequence. Preferably, said elicitor is a fungal elicitor that can beprepared by conventional means; see, e.g., Ayers, Plant Physiol. 57(1976), 760-765; Grosskopf, J. Plant Physiol. 138 (1991), 741-746;Kombrink, Plant Physiol. 81 (1986), 216-221; West, Naturwissenschaften68 (1981), 447-457.

The term “minimal promoter”, within the meaning of the present inventionrefers to nucleotide sequences necessary for transcription initiation,i.e. RNA polymerase binding, and may also include, for example, the TATAbox.

The term “pathogen” includes, for example, bacteria, viruses, fungi andprotozoa as well as elicitors prepared therefrom.

In accordance with the present invention a number of cis-acting elementshave been identified that alone are sufficient to direct high levelfungal elicitor-specific expression and that can be used to constructnovel synthetic promoters that for the first time meet the requirementsfor engineering disease resistant crops.

Studies that have been performed in accordance with the presentinvention employed a homologous transient expression system that usesparsley (Petroselinum crispum) protoplasts derived from cultured cells.This system is one of very few where the protoplasts respond to fungalelicitor molecules in an almost identical way to cells in the intactplant (Dangl, EMBO J. 6 (1987), 2551-2556; Hahlbrock, Proc. Natl. Acad.Sci. USA 92 (1995), 4150-4157). This allows the study ofelicitor-responsive cis-acting elements, something that is difficult inmany other experimental systems.

Eleven cis-acting elicitor-responsive elements (SEQ ID NOS: 3 to 13)were identified in accordance with the present invention. Monomers andmultimers of each element were constructed in addition to syntheticpromoters consisting of two or more of these elements in combination.Each construct was synthesized with either BamHI ends or with a SpeIsite at the 5′ end and an XbaI site at the 3′ end and then cloned intothe corresponding restriction site in front of a minimal CaMV 35Spromoter (−46 to +8) in the vector MS23-pBT10-GUS (Sprenger, Ph.D.thesis, University of Köln, Köln, Germany (1997); see FIG. 1 (SEQ ID NO:17) and FIG. 2). The distance between the insertion site and the TATABox varied between 25 and 70 bp depending on the insertion site employedand only slight differences, if any, were seen when the same element wasinserted into different restriction sites.

Additionally, the cis-acting elicitor-responsive element Box E17 (SEQ IDNO: 15) was identified in accordance with the present invention.Synthetic promoters were constructed comprising a monomer, a dimer orthe reverse complement of this element. Various distances between 5 and131 bp from the inserted Box E17 to the minimal promoter were testedusing monomers and dimers (see Example 7). Usable inducibility in thesense of the present invention was obtained for distances of at least 12bp, and optimal inducibility for distances of 40 to 60 bp to the 5′-endof the minimal promoter. Another cis-acting element of the presentinvention, the 21 bp long 3′-fragment of Box E17 (SEQ ID NO: 16) confersa similar elicitor-responsiveness as compared to Box E17 (see Example6).

The experiments performed in accordance with the present inventiondemonstrate that the cis-acting elements direct pathogen-inducedexpression in vivo, being active as monomers, multimers and incombination with each other within synthetic promoters. They thereforemeet the biotechnological requirements for the engineering of diseaseresistance.

In accordance with the present invention these novel chimeric promoterscloned in front of the GUS coding region and the resulting chimericgenes were introduced by means of vacuum infiltration mediated genetransfer into Arabidopsis plants; see Example 8. The expression patternobserved in the transgenic plants containing the GUS marker gene underthe control of the chimeric promoter of the invention revealedexpression in tissue infected by bacterial (Pseudomonas syringae) aswell as by fungal pathogens (Peronospora parasitica), whereas localexpression in wounded tissues seems to be inactive.

The chimeric promoter of the invention may be preferably comprised onlyof the above defined cis-acting elements and a minimal promoter. As willbe discussed below, other regulatory sequences may be added or presentdependent on the intended use of the chimeric promoter of the invention.However, preferably the chimeric promoter of the invention lackselements that interfere with the elicitor specific expression and/orwhich are responsible for the non-selective expression of the promoterthe cis-acting element of the invention was derived from.

To obtain possible expression in all tissues of a transgenic plant, theminimal regulatory sequences of constitutive promoters are often used,such as the 35 S promoter of CaMV (Odell, Nature 313 (1985), 810-812) orpromoters of the polyubiquitin genes of maize (Christensen, Plant Mol.Biol. 18 (1982), 675-689). It is also immediately evident to the personskilled in the art that further regulatory elements may be added to thechimeric sequences of the invention. For example, transcriptionalenhancers and/or sequences which allow for further induced expression ofthe chimeric promoter of the invention may be employed. Enhancersequences functional in plants include, for example, ocs-element (Ellis,EMBO J. 6 (1987), 3203-3208); the family of ACGT-elements (hex-motif,G-box as 1-element) (Williams, Plant Cell 4 (1992), 485-496) and thecyt-1 element (Neuteboom, Plant J. 4 (1993), 525-534). In order toachieve expression in specific tissues of a transgenic plant it ispossible to use tissue specific promoters (see, e.g., Stockhaus, EMBO J.8 (1989), 2245-2251). Known are also promoters which are specificallyactive in tubers of potatoes or in seeds of different plants species,such as maize, Vicia, wheat, barley etc. Furthermore, the chemicallyinducible Tet-system may be employed (Gatz, Mol. Gen. Genet. 227 (1991);229-237). Further suitable promoters are known to the person skilled inthe art and are described, e.g., in Ward (Plant Mol. Biol. 22 (1993),361-366).

Preferably, the chimeric promoter of the invention further comprises acis-acting element having the nucleotide sequence of SEQ ID NO: 1 or 2;see Example 5.

In a particularly preferred embodiment of the invention the chimericpromoter comprises homo- and/or hetero-multimeric forms of saidcis-acting element(s); see also the appended Example 5. Preferably, saidmultimeric form is a dimer or tetramer. Particularly preferred are thosecombinations of cis-acting elements that are described in Example 5 andwhich combination provide for an at least 20-fold, preferably at least30-fold and particularly preferred at least about 50-fold induction.

In a preferred embodiment of the chimeric promoter of the invention theminimal promoter is derived from the CaMV35S promoter, CHS promoter, PR1promoter, or hcbt2 promoter. However, other minimal promoters from othersources may be employed as well.

In a further preferred embodiment of the chimeric promoter of theinvention, the distance between said cis-acting element and said minimalpromoter is 12 to 300 base pairs, more preferably 25 to 70 base pairs,and most preferably 40 to 60 base pairs. In addition or alternatively, aspacer region preferably composed of 4 to 10 base pairs separates atleast two of said cis-acting elements in the chimeric promoter.Likewise, it is preferred that at least two of said multimeric forms inthe chimeric promoter described above are separated by a spacer ofbetween about 50 to 1000 base pairs.

In a particularly preferred embodiment of the chimeric promoter of theinvention the induction of gene expression upon elicitor treatment orpathogen infection is at least 15-fold. As discussed before, thecis-acting elements so far investigated in the prior art only providedfor induction upon elicitor treatment of about 10-fold. However, a10-fold induction of a recombinant gene encoding, e.g., an anti-viralprotein may not be sufficient to rapidly and efficiently combat againstthe pathogen. The present invention provides several cis-acting elementsthat are capable of inducing high level expression of a given DNAsequence up to 400-fold induction; see, e.g., Example 1. Furthermore,the invention demonstrates that the combination of otherwise weakcis-acting elements can provide for a substantial increase of theoverall inducibility of the chimeric promoter; see Example 5. Thus, thepresent invention for the first time provides a generally applicablemethod for how to construct and use chimeric promoters in the field ofplant biotechnology. As will be noted from the appended Examples, thebackground value of the chimeric promoters of the invention may vary toa certain extent. The person skilled in the art therefore may employdifferent chimeric promoters with different background levels andinducibility depending on the intended use. For example, if the approachof coat protein-mediated protection against virus infection is used thechimeric promoter employed may have high background level expressionthat would not harm the plant and which upon viral infection wouldincrease at high levels such that resistance to the virus can beobtained. The same rational would apply to, e.g., an antisense orribozyme mediated protection or the engineering of resistance to fungalpathogens by the expression of anti-fungal proteins etc. On the otherhand, where the generation of race-specific resistant genes andartificial generation of hypersensitive cell death is intended,preferably a chimeric promoter is used that has low or substantially nobackground activity and that only upon pathogen attack is activated toan extent that sufficient level of toxic protein is made so as to causethe cell to die. The selection of the appropriate chimeric promoter ofthe invention depending on its use is well within the skill of theperson skilled in the art.

Examples of the different possible applications of the chimeric promoteraccording to the invention as well as its cis-acting elements will bedescribed in detail in the following.

Hence, in a further embodiment, the present invention relates to arecombinant gene comprising the above-described chimeric promoter.Preferably, the recombinant gene is configured such that the chimericpromoter is operatively linked to a heterologous DNA sequence.

The term “heterologous” with respect to the DNA sequence beingoperatively linked to the chimeric promoter of the invention means thatsaid DNA sequence is not naturally linked to the chimeric promoter ofthe invention.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. The chimeric promoter “operablylinked” to a heterologous DNA sequence is ligated in such a way thatexpression of a coding sequence is achieved under conditions compatiblewith the control sequences. Expression comprises transcription of theheterologous DNA sequence preferably into a translatable mRNA.Regulatory elements ensuring expression in eukaryotic, i.e. plant cellsare well known to those skilled in the art. In the case of eukaryoticcells they comprise optionally poly-A signals ensuring termination oftranscription and stabilization of the transcript, for example, those ofthe 35S RNA from Cauliflower Mosaic Virus (CaMV) and the NopalineSynthase gene from Agrobacterium tumefaciens. Additional regulatoryelements may include transcriptional as well as translational enhancers.A plant translational enhancer often used is the CAMV omega sequences,the inclusion of an intron (Intron-1 from the Shrunken gene of maize,for example) has been shown to increase expression levels by up to100-fold. (Mait, Transgenic Research 6 (1997), 143-156; Ni, PlantJournal 7 (1995), 661-676). In this respect, it should be noted that inone embodiment of the recombinant gene of the invention at least one ofsaid cis-acting elements is located in the 5′- or 3-untranslated regionor in an intron of the recombinant gene.

In a preferred embodiment of the recombinant gene of the invention saidheterologous DNA sequence encodes a (poly)peptide, cytotoxic protein,antibody, antisense RNA, sense RNA, ribozyme, transcription factor,protease, nuclease, lipase, or polymerase.

The recombinant gene of the invention can be used alone or as part of avector to express heterologous DNA sequences, which, e.g., encodeproteins for, e.g., the control of disease resistance or diagnostics ofpathogen inducible or related gene expression. The recombinant gene orvector containing the DNA sequence encoding an RNA or a protein ofinterest is introduced into the cells which in turn produce the RNA orprotein of interest. For example, the chimeric promoter of the inventioncan be operatively linked to DNA sequences encoding Barnase for use inthe production of localized cell death in plants upon pathogen attack.

On the other hand, said protein can be a scorable marker, e.g.,luciferase, green fluorescent protein or β-galactosidase. Thisembodiment is particularly useful for simple and rapid screening methodsfor compounds and substances described herein below capable ofmodulating pathogene specific or elicitor inducible gene expression. Forexample, transgenic plant cells can be cultured in the presence andabsence of a candidate compound in order to determine whether thecompound affects the expression of genes which are under the control ofchimeric promoters of the invention, which can be measured, e.g., bymonitoring the expression of the above-mentioned marker. It is alsoimmediately evident to those skilled in the art that other marker genesmay be employed as well, encoding, for example, a selectable markerwhich provides for the direct selection of compounds which induce orinhibit the expression of said marker.

The chimeric promoters of the invention may also be used in methods ofantisense approaches. The antisense RNA may be a short (generally atleast 10, preferably at least 14 nucleotides, and optionally up to 100or more nucleotides) nucleotide sequence formulated to be complementaryto a portion of a specific mRNA sequence and/or DNA sequence of the geneof interest. Standard methods relating to antisense technology have beendescribed; see, e.g., Klann, Plant Physiol. 112 (1996), 1321-1330.Following transcription of the DNA sequence into antisense RNA, theantisense RNA binds to its target sequence within a cell, therebyinhibiting translation of the mRNA and down-regulating expression of theprotein encoded by the mRNA.

Furthermore, appropriate ribozymes can be employed (see, e.g., EP-A1 0291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave the(pre)-mRNA of a target gene. Selection of appropriate target sites andcorresponding ribozymes can be done as described for example inSteinecke, Ribozymes, Methods in Cell Biology 50, Galbraith, edsAcademic Press, Inc. (1995), 449-460. Further applications of thechimeric promoter are evident to the person skilled in the art and canbe derived from the literature, e.g., Strittmatter and Wegener,Zeitschrift für Naturforschung 48c (1993), 673-688; Kahl, J. Microbiol.Biotechnol. 11 (1995), 449-460 and references cited therein.

Said transcription factor can for example be a master regulatory factorthat controls the expression of a cascade of genes involved in pathogendefense of the plant (Grotewold, Plant Cell 10 (1998), 721-740; Rushtonand Somssich, Curr. Opin. Plant Biol. 1 (1998), 311-315). Alternatively,it can be a hybrid transcription factor containing a DNA-binding domain(e.g. of GAL4 or of the bacteriophage 434) and an activator domain (e.g.of VP 16 or of any functional plant activator domain), which, whenexpressed in transgenic plants containing an antisense target gene underthe control of a synthetic promoter containing the appropriatecis-acting element recognizing the hybrid factor, leads to specificrepression (knock-out) of the desired endogenous gene function (Wilde,Plant Mol. Biol. 24 (1994), 381-388; Guyer, Genetics 149 (1998),633-639).

Suitable lipases comprise for example phospholipases, e.g., C or A₂ typephospholipases (Scherer, Plant Growth regulation 18 (1996), 125-133).Lipases are capable of releasing free fatty acids from membrane lipids,wherein these fatty acids can function as signal transducers by whichgeneral cellular defense reactions are elicited. The growing importanceof free fatty acids in pathogen-defense is documented, e.g., in Scherer(1996), Roy (Plant Sci. 107 (1995), 17-25 and references cited therein)and Tavernier (Plant Sci. 104 (1995), 117-125).

Nucleases, i.e. RNases and DNases, may also be employed, of whichBarnase is one candidate among others. The use of proteases in thecontext of this embodiment may apply to cytotoxic effects.

A signal amplification system may be constructed using polymerases. In atwo-step model, an elicitor-induced polymerase, e.g., SP6-, T7- orT3-RNA polymerase, can transcribe a second recombinant gene which iscontrolled by a promoter to which the polymerase is highly specific. Thesecond gene may encode for example a cytotoxic protein which is thenexpressed in an amplified way. A plant system based on T7-RNA polymerasewas described by McBride (Proc. Natl. Acad. Sci. USA 91 (1994),7301-7305).

Cytotoxic proteins comprise, for example, plant RIPs (ribosomeinactivating proteins; (Stripe, Bio/Technology 10 (1992), 405-412),defensins (Broekaert, Plant Physiol. 108 (1995), 1353-1358), Bt toxin,α-amylase inhibitor, T4-lysozyme, avirulence gene products, or enzymessuch as glucose oxidase which generate reactive oxygen species (Shah,Trends Biotechnol. 13 (1995), 362-368; Shah, Curr. Opin. Biotech. 8(1997), 208-214; Beachy, Curt Opin. Biotech. 8 (1997), 215-220;Cornelissen, Plant Physiol. 101 (1993), 709-712; Estruch, Nucleic AcidsRes. 22 (1994), 3983-3989).

It is in principle possible to modify the coding sequence in such a waythat the protein is located in any desired compartment of the plantcell. These include the nucleus, endoplasmatic reticulum, the vacuole,the mitochondria, the plastids, the apoplast, the cytoplasm etc. Methodshow to carry out this modifications and signal sequences ensuringlocalization in a desired compartment are well known to the personskilled in the art. (Görlich, Science 271 (1996), 1513-1518; Hicks,Plant Physiol. 107 (1995), 1055-1058; Rachubinski, Cell 83 (1995),525-528; Schatz, Science 271 (1996), 1519-1526; Schnell, Cell 83 (1995),521-524; Verner, Science 241 (1988), 1307-1313; Vitale, BioEssays 14(1992), 151-160).

The present invention also relates to vectors, particularly plasmids,cosmids, viruses and bacteriophages used conventionally in geneticengineering that comprise a chimeric promoter or a recombinant gene ofthe invention. Preferably, said vector is a plant expression vector,preferably further comprising a selection marker for plants. For exampleof suitable selector markers, see supra. Methods which are well known tothose skilled in the art can be used to construct recombinant vectors;see, for example, the techniques described in Sambrook, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.and Ausubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1989). Alternatively, thechimeric promoters and recombinant genes of the invention can bereconstituted into liposomes for delivery to target cells.

Advantageously, the above-described vectors of the invention comprise aselectable and/or scorable marker. Selectable marker genes useful forthe selection of transformed plant cells, callus, plant tissue andplants are well known to those skilled in the art and comprise, forexample, antimetabolite resistance as the basis of selection for dhfr,which confers resistance to methotrexate (Reiss, Plant Physiol. (LifeSci. Adv.) 13 (1994), 143-149); npt, which confers resistance to theaminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella,EMBO J. 2 (1983), 987-995) and hygro, which confers resistance tohygromycin (Marsh, Gene 32 (1984), 481-485). Additional selectable geneshave been described, namely trpB, which allows cells to utilize indolein place of tryptophan; hisD, which allows cells to utilize histinol inplace of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988),8047); mannose-6-phosphate isomerase which allows cells to utilizemannose (WO 94/20627) and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.)or deaminase from Aspergillus terreus which confers resistance toBlasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995),2336-2338).

Useful scorable marker are also known to those skilled in the art andare commercially available. Advantageously, said marker is a geneencoding luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J.Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett.389 (1996), 44-47) or β-glucuronidase (Jefferson, EMBO J. 6 (1987),3901-3907). This embodiment is particularly useful for simple and rapidscreening of cells, tissues and plants containing a vector of theinvention.

The present invention furthermore relates to host cells comprising achimeric promoter, recombinant gene or a vector according to theinvention wherein the chimeric promoter is foreign to the host cell.

By “foreign” it is meant that the chimeric promoter is eitherheterologous with respect to the host cell, this means derived from acell or organism with a different genomic background, or is homologouswith respect to the host cell but located in a different genomicenvironment than the naturally occurring counterpart of said cis-actingelement. This means that, if the cis-acting element is homologous withrespect to the host cell, it is not located in its natural location inthe genome of said host cell, in particular it is surrounded bydifferent genes. The vector or recombinant gene according to theinvention which is present in the host cell may either be integratedinto the genome of the host cell or it may be maintained in some formextrachromosomally. In this respect, it is also to be understood thatthe chimeric promoter or recombinant gene of the invention can be usedto restore or create a mutant gene via homologous recombination(Paszkowski (ed.), Homologous Recombination and Gene Silencing inPlants. Kluwer Academic Publishers (1994)). The host cell can be anyprokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plantor animal cells. Preferred cells are plant cells.

In a further preferred embodiment, the present invention provides for amethod for the production of transgenic plants, plant cells or planttissue comprising the introduction of a chimeric promoter, recombinantgene or vector of the invention into the genome of said plant, plantcell or plant tissue. For the expression of the heterologous DNAsequence under the control of the chimeric promoter according to theinvention in plant cells, further regulatory sequences such as poly Atail may be fused, preferably 3′ to the heterologous DNA sequence, seealso supra. Further possibilities might be to add Matrix AttachmentSites at the borders of the transgene to act as “delimiters” andinsulate against methylation spread from nearby heterochromaticsequences.

Methods for the introduction of foreign genes into plants are also wellknown in the art. These include, for example, the transformation ofplant cells or tissues with T-DNA using Agrobacterium tumefaciens orAgrobacterium rhizogenes, the fusion of protoplasts, direct genetransfer (see, e.g., EP-A 164 575), injection, electroporation, vacuuminfiltration, biolistic methods like particle bombardment,pollen-mediated transformation, plant RNA virus-mediated transformation,liposome-mediated transformation, transformation using wounded orenzyme-degraded immature embryos, or wounded or enzyme-degradedembryogenic callus and other methods known in the art. The vectors usedin the method of the invention may contain further functional elements,for example “left border”- and “right border”-sequences of the T-DNA ofAgrobacterium which allow stable integration into the plant genome.Furthermore, methods and vectors are known to the person skilled in theart which permit the generation of marker free transgenic plants, i.e.the selectable or scorable marker gene is lost at a certain stage ofplant development or plant breeding. This can be achieved by, forexample cotransformation (Lyznik, Plant Mol. Biol. 13 (1989), 151-161;Peng, Plant Mol. Biol. 27 (1995), 91-104) and/or by using systems whichutilize enzymes capable of promoting homologous recombination in plants(see, e.g., WO97/08331; Bayley, Plant Mol. Biol. 18 (1992), 353-361);Lloyd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet.230 (1991), 170-176; Onouchi, Nucl. Acids Res. 19 (1991), 6373-6378).Methods for the preparation of appropriate vectors are described by,e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition(1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Suitable strains of Agrobacterium tumefaciens and vectors as well astransformation of Agrobacteria and appropriate growth and selectionmedia are well known to those skilled in the art and are described inthe prior art (GV3101 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986),383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777;Bevan, Nucleic. Acid Res. 12 (1984), 8711; Koncz, Proc. Natl. Acad. Sci.USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol. 20 (1992), 963-976;Koncz, Specialized vectors for gene tagging and expression studies. In:Plant Molecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.),Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-120516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4,1-46; An, EMBO J. 4 (1985), 277-287). Although the use of Agrobacteriumtumefaciens is preferred in the method of the invention, otherAgrobacterium strains, such as Agrobacterium rhizogenes, may be used,for example if a phenotype conferred by said strain is desired.

Methods for the transformation using biolistic methods are well known tothe person skilled in the art; see, e.g., Wan, Plant Physiol. 104(1994), 37-48; Vasil, Bio/Technology 11 (1993), 1553-1558 and Christou(1996) Trends in Plant Science 1, 423-431. Microinjection can beperformed as described in Potrykus and Spangenberg (eds.), Gene TransferTo Plants. Springer Verlag, Berlin, N.Y. (1995).

The transformation of most dicotyledonous plants is possible with themethods described above. But also for the transformation ofmonocotyledonous plants several successful transformation techniqueshave been developed. These include the transformation using biolisticmethods as, e.g., described above as well as protoplast transformation,electroporation of partially permeabilized cells, introduction of DNAusing glass fibers, etc.

The resulting transformed plant cell can then be used to regenerate atransformed plant in a manner known by a skilled person.

Alternatively, a plant cell can be used and modified such that saidplant cell expresses an endogenous gene under the control of thechimeric promoter. The introduction of the chimeric promoter of theinvention which does not naturally control the expression of a givengene or genomic sequences using, e.g., gene targeting vectors can bedone according to standard methods, see supra and, e.g., Hayashi,Science 258 (1992), 1350-1353; Fritze and Walden, Gene activation byT-DNA tagging. In Methods in Molecular biology 44 (Gartland, K. M. A.and Davey, M. R., eds). Totowa: Human Press (1995), 281-294) ortransposon tagging (Chandlee, Physiologia Plantarum 78 (1990), 105-115).

In general, the plants which can be modified according to the inventioncan be derived from any desired plant species. They can bemonocotyledonous plants or dicotyledonous plants, preferably they belongto plant species of interest in agriculture, wood culture orhorticulture interest, such as crop plants (e.g. maize, rice, barley,wheat, rye, oats etc.), potatoes, oil producing plants (e.g. oilseedrape, sunflower, pea nut, soy bean, etc.), cotton, sugar beet, sugarcane, leguminous plants (e.g. beans, peas etc.), wood producing plants,preferably trees, etc.

Thus, the present invention relates also to transgenic plant cellscomprising, preferably stably integrated into the genome, a chimericpromoter, a recombinant gene or vector according to the invention orobtainable by the above-described method.

Furthermore, the present invention also relates to transgenic plants andplant tissue comprising the above-described transgenic plant cells orobtainable by the above-described method. These plants may show, forexample, increased disease resistance.

In a preferred embodiment of the invention, the transgenic plant uponthe presence of the chimeric promoter or the recombinant gene of theinvention attained resistance or improved resistance against a pathogenthe corresponding wild-type plant was susceptible to.

The term “resistance” covers the range of protection from a delay tocomplete inhibition of disease development. Examples for pathogens ofimportance comprise Phytophthora infestans, the causal agent of potatolate blight disease, Phytophthora sojae, root rot pathogen of soybean,Peronospora parasitica (downy mildew), Magnaporthe grisea, causal agentof rice blast disease, Erysiphe spp (powdery mildew), Pseudomonassyringae (agent of bacterial blight), Enwinia amylovora (fire blightdisease), Erwinia carotovora (soft rot), Botrytis cinerea (downy mildewof grape), Rhizoctonia solani and Pythium debaryanum (agents of seedlingblight or damping off disease).

In yet another aspect the invention also relates to harvestable partsand to propagation material of the transgenic plants according to theinvention which contain transgenic plant cells described above.Harvestable parts can be in principle any useful part of a plant, forexample, leaves, stems, fruit, seeds, roots etc. Propagation materialincludes, for example, seeds, fruits, cuttings, seedlings, tubers,rootstocks etc.

As discussed above, novel cis-acting elements have been identified inaccordance with the present invention that are capable of conferringelicitor inducible or pathogen specific gene expression in plant cellsand plants. Therefore, the present invention also relates to cis-actingelements as defined above or multimeric forms of any one of those asdiscussed hereinbefore.

Due to the tight regulation of the chimeric promoters of the inventionit is evident that they are particularly suited for the identificationof compounds that either specifically interact with these cis-actingelements or that act upstream of the signal transduction pathway thatleads to activation of genes the cis-acting elements were derived from.

Thus, the present invention further relates to a method for theidentification of an activator or inhibitor of genes specificallyexpressed in plants upon pathogen infection comprising the steps of:

(a) providing a plant, plant cell, or plant tissue comprising arecombinant DNA molecule comprising a readout system operatively linkedto the chimeric promoter of the invention;

(b) culturing said plant cell or tissue or maintaining said plant in thepresence of a compound or a sample comprising a plurality of compoundsunder conditions which permit expression of said readout system;

(c) identifying or verifying a sample and compound, respectively, whichleads to suppression or activation and/or enhancement of expression ofsaid readout system in said plant, plant cell, or plant tissue.

For the identification of inhibitors, it is advantageous to include anelicitor or another activator known to be capable of inducing theactivity of promoters that contain the cis-acting elements of thechimeric promoters of the invention in step (b) of the above-describedmethod, and to determine whether the compound to be screened suppressesthe induction of the readout system by said elicitor or activator.

The term “readout system” in context with the present invention means aDNA sequence which upon transcription and/or expression in a cell,tissue or organism provides for a scorable and/or selectable phenotype.Such readout systems are well known to those skilled in the art andcomprise, for example, recombinant genes and marker genes as describedabove and in the appended examples.

The term “plurality of compounds” in a method of the invention is to beunderstood as a plurality of substances which may or may not beidentical.

Said compound or plurality of compounds may be comprised in, forexample, samples of inorganic or organic molecules or, e.g., cellextracts from, e.g., plants, animals or microorganisms. Furthermore,said compound(s) may be known in the art but hitherto not known to becapable of suppressing or activating pathogen related genes. Suitableset ups for the method of the invention are known to the person skilledin the art. The plurality of compounds may be, e.g., added to the cellor tissue culture medium or soil, injected into the cell or sprayed ontothe plant.

If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is either possible toisolate the compound from the original sample identified as containingthe compound capable of suppressing or activating the chimeric promoterof the invention, or one can further subdivide the original sample, forexample, if it consists of a plurality of different compounds, so as toreduce the number of different substances per sample and repeat themethod with the subdivisions of the original sample. Depending on thecomplexity of the samples, the steps described above can be performedseveral times, preferably until the sample identified according to themethod of the invention only comprises a limited number of or only onesubstance(s). Preferably said sample comprises substances of similarchemical and/or physical properties, and most preferably said substancesare identical. Preferably, the compound identified according to theabove described method or its analog or derivative is further formulatedin a form suitable for the application in plant breeding or plant celland tissue culture. For example, it can be combined with aagriculturally acceptable carrier known in the art.

The compounds which can be tested and identified according to a methodof the invention may be expression libraries, e.g., cDNA expressionlibraries, peptides, proteins, nucleic acids, antibodies, small organiccompounds, hormones, peptidomimetics, PNAs or the like (Milner, NatureMedicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell79 (1994), 193-198 and references cited supra). Furthermore, genesencoding a putative regulator of genes controlled by the cis-actingelements of the invention and/or which exert their effects up- ordownstream from such genes may be identified using, for example,insertion mutagenesis using, for example, gene targeting vectors knownin the art (see, e.g., Hayashi, Science 258 (1992), 1350-1353; Fritzeand Walden, Gene activation by T-DNA tagging. In Methods in Molecularbiology 44 (Gartland, K. M. A. and Davey, M. R., eds). Totowa: HumanPress (1995), 281-294) or transposon tagging (Chandlee, PhysiologiaPlantarum 78 (1990), 105-115).

Said compounds can also be functional derivatives or analogues of knowninhibitors or activators. Methods for the preparation of chemicalderivatives and analogues are well known to those skilled in the art andare described in, for example, Beilstein, Handbook of Organic Chemistry,Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010U.S.A. and Organic Synthesis, Wiley, N.Y., USA. Furthermore, saidderivatives and analogues can be tested for their effects according tomethods known in the art or as described in the appended examples.Furthermore, peptidomimetics and/or computer aided design of appropriatederivatives and analogues can be used. The cell or tissue that may beemployed in the method of the invention preferably is a plant cell,plant tissue or plant of the invention described in the embodimentshereinbefore.

In an additional embodiment, the characteristics of a given compound maybe compared to that of a cell contacted with a compound which is eitherknown to be capable or incapable of suppressing or activating thechimeric promoter of the invention or the promoter the cis-actingelement of the chimeric promoter is derived from.

The inhibitor or activator identified by the above-described method mayprove useful as a plant protective agent or herbicide or pesticide.Thus, in a further embodiment the invention relates to a compoundobtained or identified according to the method of the invention saidcompound being an activator or an inhibitor of genes specificallyinduced upon pathogen infection.

Furthermore, identification of trans-acting factors which interact withthe cis-acting elements of the invention can form the basis for thedevelopment of novel agents for modulating conditions associated withplant diseases. Identification of trans-acting factors is carried outusing standard methods in the art (see, e.g., Sambrook, supra, andAusubel, supra). To determine whether a protein binds to the cis-actingelements of the invention standard DNA footprinting and/or nativegel-shift analyses can be carried out. In order to identify thetrans-acting factor which binds to the cis-acting elements of theinvention, these elements can be used as an affinity reagent in standardprotein purification methods, or as a probe for screening an expressionlibrary. Once the trans-acting factor is identified, modulation of itsbinding to the cis-acting elements of the invention can be pursued,beginning with, for example, screening for inhibitors of trans-actingfactor binding.

Activation or repression of genes involved in plant defense reactionscould then be achieved in plants by applying of the trans-acting factor(or its inhibitor) or the gene encoding it, e.g. in a vector fortransgenic plants. In addition, if the active form of the trans-actingfactor is a dimer, dominant-negative mutants of the trans-acting factorcould be made in order to inhibit its activity. Furthermore, uponidentification of the trans-acting factor, further components in thepathway leading to activation (e.g. signal transduction) or repressionof pathogenesis related genes then can be identified. Modulation of theactivities of these components can then be pursued, in order to developadditional agents and methods for modulating the response of plants uponpathogen attack in plants.

Accordingly, the present invention also relates to a plant protectioncomposition comprising the compound identified and obtained by the abovedescribed methods. The plant protection composition can be prepared byemploying the above-described method of the invention and synthesizingthe compound identified as inhibitor or activator in an amountsufficient for use in agriculture. Thus, the present invention alsorelates to a method for the preparation of an agricultural plantprotection composition comprising the above-described steps of themethod of the invention and synthesizing the compound so identified oran analog or derivative thereof.

In the plant protection composition of the invention, the compoundidentified by the above-described method may be preferentiallyformulated by conventional means commonly used for the application of,for example, herbicides and pesticides or agents capable of inducingsystemic acquired resistance (SAR). For example, certain additives knownto those skilled in the art stabilizers or substances which facilitatethe uptake by the plant cell, plant tissue or plant may be used, forexample, harpins, elicitins, salicylic acid (SA),benzol(1,2,3)thiadiazole-7-carbothioic acid (BTH), 2,6-dichloroisonicotinic acid (INA), jasmonic acid (JA), methyljasmonate.

In a further embodiment, the present invention relates to an antibodyspecifically recognizing the compound obtainable by the method of theinvention or the cis-acting element described above. The antibodies ofthe invention can be used to identify and isolate other activators andinhibitors of genes that are involved in plant defense. These antibodiescan be monoclonal antibodies, polyclonal antibodies or syntheticantibodies as well as fragments of antibodies, such as Fab, Fv or scFvfragments etc. Monoclonal antibodies can be prepared, for example, bythe techniques as originally described in Köhler and Milstein, Nature256 (1975), 495, and Galfré, Meth. Enzymol. 73 (1981), 3, which comprisethe fusion of mouse myeloma cells to spleen cells derived from immunizedmammals. Furthermore, antibodies or fragments thereof to theaforementioned peptides can be obtained by using methods which aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor, 1988.

Furthermore, the present invention relates to a diagnostic compositioncomprising the chimeric promoter, the recombinant gene, the vector, thecompound or the antibody of the invention, and optionally suitable meansfor detection. Said diagnostic compositions may be used for, e.g.,methods for screening activators or inhibitors as described above.

In addition, the present invention relates to a kit comprising thechimeric promoter, the recombinant gene, the vector, the compound or theantibody of the invention. The kit of the invention may contain furtheringredients such as selection markers and components for selective mediasuitable for the generation of transgenic plant cells, plant tissue orplants. Furthermore, the kit may include buffers and substrates forreporter genes that may be present in the recombinant gene or vector ofthe invention. In addition, the kit of the invention may containcompounds such as elicitors, preferably fungal elicitors that can beused as standards for the expression assays. The kit of the inventionmay advantageously be used for carrying out the method of the inventionand could be, inter alia, employed in a variety of applications referredto herein, e.g., in the diagnostic field or as research tool. The partsof the kit of the invention can be packaged individually in vials or incombination in containers or multicontainer units. Manufacture of thekit follows preferably standard procedures which are known to the personskilled in the art.

The kit or its ingredients according to the invention can be used inplant cell and plant tissue cultures, for example, for any of the abovedescribed methods for detecting inhibitors and activators ofpathogenesis related genes. The kit of the invention and its ingredientsare expected to be very useful in breeding new varieties of, forexample, plants which display improved properties such as diseaseresistance.

It is also immediately evident to the person skilled in the art that thechimeric promoters, recombinant genes and vectors of the presentinvention can be employed to produce transgenic plants with a desiredtrait (see for review TIPTEC Plant Product & Crop Biotechnology 13(1995), 312-397) comprising (i) insect resistance (Vaek, Plant Cell 5(1987), 159-169), (ii) virus resistance (Powell, Science 232 (1986),738-743; Pappu, World Journal of Microbiology & Biotechnology 11 (1995),426-437; Lawson, Phytopathology 86 (1996), 56 suppl.), (iii) resistanceto bacteria, insects and fungi (Duering, Molecular Breeding 2 (1996),297-305; Strittmatter, Bio/Technology 13 (1995), 1085-1089; Estruch,Nature Biotechnology 15 (1997), 137-141), (iv) inducing and maintainingmale and/or female sterility (EP-A1 0 412 006; EP-A1 0 223 399;WO93/25695) or may be used as highly inducible production systems ofheterologous proteins or biopolymers in plants analogous to induciblesystems in bacteria.

The present invention for the first time demonstrates that a number ofcis-acting elements that are responsible for inducibility ofpathogenesis-related genes can be used either alone or in combinationwith themselves or with other cis-acting elements to construct chimericpromoters that are capable of mediating highly inducible gene expressionin plant cells upon elicitor treatment. It is therefore evident thatcis-acting elements derived, e.g., from pathogen-related promoters otherthan those specifically described above can be used in accordance withthe present invention, for example, chitinase promoters; see, e.g.,Kellmann, Plant. Mol. Biol. 30 (1996), 351-358. Appropriate promotersthat provide a source for such cis-acting elements can be used andobtained from any plant species, for example, maize, potato, sorghum,millet, coix, barley, wheat and rice. Such promoters are characterizedby their inducibility upon pathogen infection.

For example, using cDNA of proteins that are specifically expressed inplants upon pathogen attack as probes, a genomic library consisting ofplant genomic DNA cloned into phage or bacterial vectors can be screenedby a person skilled in the art. Such a library consists, e.g., ofgenomic DNA prepared from plant leaf tissue, fractionized in fragmentsranging from 5 kb to 50 kb, cloned into the lambda vectors such asLambda EMBL3 or 4, Lambda ZAP, Lambda DASH or Lambda GEM. Phageshybridizing with the probes can be purified. From the purified phagesDNA can be extracted and sequenced. Having isolated the genomicsequences corresponding to the genes encoding the PR proteins, it ispossible to fuse heterologous DNA sequences to these promoters or theirregulatory sequences via transcriptional or translational fusionsaccording to methods well known to the person skilled in the art. Inorder to identify the regulatory sequences and specific elements of thethese genes, 5′-upstream genomic fragments can be cloned in front ofmarker genes such as luc, gfp or the GUS coding region and the resultingchimeric genes can be introduced by means of Agrobacterium tumefaciensmediated gene transfer into plants or transfected into plant cells orplant tissue for transient expression. The expression pattern observedin the transgenic plants or transfected plant cells containing themarker gene under the control of the isolated regulatory sequencesreveal the boundaries of the promoter and its cis-acting elements. Theisolation of cis-acting elements having the above defined properties canbe done by conventional techniques known in the art, for example, byusing DNAseI footprinting and loss-and gain-of-function experiments. Itis then possible to isolate the corresponding promoter region byconventional techniques and test it for its expression pattern. For thispurpose, it is, for instance, possible to fuse the putative cis-actingelement with a minimal promoter to a reporter gene, such as GUS,luciferase or green fluorescent protein (GFP) and assess the expressionof the reporter gene in transient expression assays or transgenicplants; see also the appended examples.

Thus, the present invention relates to the use of a cis-acting elementsufficient to direct elicitor-specific expression and in particular tothe use of the chimeric promoter, the recombinant gene, the vector, thecis-acting element and/or the compound of the present invention for theproduction of pathogen resistant plants or for identifying and/orproducing compounds capable of conferring induced resistance to apathogen in a plant.

In a still further embodiment, the present invention relates to a methodof rendering a gene responsive to pathogens comprising inserting atleast one cis-acting element sufficient to direct elicitor-specificexpression into the promoter of said gene. As is evident to the personskilled in the art a promoter that displays the capabilities of thechimeric promoter of the invention can also be obtained by introducingthe cis-acting element as defined above into a promoter of a gene,preferably in close proximity to the transcription initiation site ofthe gene.

In another embodiment, the present invention relates to a method forpreparing a promoter capable of mediating local gene expression inplants upon pathogen infection comprising operably linking a cis-actingelement sufficient to direct elicitor-specific expression to atranscription initiation sequence of a promoter. Preferably, saidcis-acting element to be inserted in the above-described methods is acis-acting element of the present invention or as defined in theforegoing embodiments or a multimeric form thereof as definedhereinabove. As mentioned before, the elicitor responsive cis-actingelements are preferably responsive to fungal elicitor.

In a preferred embodiment of the invention, the above-described methodsfurther comprising deleting non-specific cis-acting elements in thepromoter. Introduction of the cis-acting element of the invention into agiven promoter per se may not be sufficient to direct the promoter toexclusively mediate local gene expression in plants upon pathogeninfection. In this case, preexisting elements that may be responsive,for example, to light, hormones, low temperatures, drought or saltstress may be deleted.

The above described methods give rise to novel chimeric promoters thatare at least partially, preferably fully controlled by plant/pathogeninteraction.

Accordingly, the present invention also relates to the promoterobtainable by a method as described above. Said promoter can then beemployed for the embodiments described hereinabove.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the methods, uses and compounds to be employed inaccordance with the present invention may be retrieved from publiclibraries, using for example electronic devices. For example the publicdatabase “Medline” may be utilized which is available on the Internet.Further databases and public website addresses are known to the personskilled in the art and can also be obtained using known websites forInternet search engines. An overview of patent information inbiotechnology and a survey of relevant sources of patent informationuseful for retrospective searching and for current awareness is given inBerks, TIBTECH 12 (1994), 352-364.

The present invention is further described by reference to the followingnon-limiting examples.

The Examples illustrate the invention:

Experimental Setup

1. Recombinant DNA Techniques

Unless stated otherwise in the examples, all recombinant DNA techniquesare performed according to protocols as described in Sambrook (1989),Molecular Cloning: A Laboratory Manual. Cold Spring Harbor LaboratoryPress, NY or in Volumes 1 and 2 of Ausubel (1994), Current Protocols inMolecular Biology, Current Protocols. Standard materials and methods forplant molecular work are described in Plant Molecular Biology Labfase(1993) by R. D. D. Croy, jointly published by BIOS ScientificPublications Ltd. (UK) and Blackwell Scientific Publications (UK).

2. Transient Expression Vector

All constructs, unless a different protocol is given in the examples,were cloned between the SpeI and XbaI sites in pbt10-GUS (ms23)(Sprenger, 1997). At the 3′ end of each construct is an intact XbaI site(6 bp) followed immediately by a minimal CaMV 35S promoter (−46 to +8).The 3′ end of all inserts are therefore 28 bp upstream of the CaMV TATABox and 52 bp upstream of the start of transcription. Multiple copies ofthe elements are separated by 6 base pairs (TCTAGT) created by theligation of a SpeI sticky end with a XbaI sticky end. The sequence ofms23 (SEQ ID NO: 1-7) as a restriction map and an overview cartoon areprovided (FIGS. 1 and 2).

3. Transgenic Plant Vector

The vector employed was pGPTV-GUS-kan (Becker, Plant Mol. Biol. 20(1992), 1195-1197). The polylinker, minimal CaMV 35S promoter and GUSreporter gene are identical to ms23. All spacings and orders ofcis-elements within the constructs are therefore identical to those inthe corresponding transient expression constructs in ms23. A cartoon ofpGPTV is provided (FIG. 3).

4. Transient Transfection and Expression Assays

The transient transfection and expression assays were essentiallycarried out as described in Dangl, EMBO J. 6 (1987), 2551-2556;Schulze-Lefert, EMBO J. 8 (1989), 651-656; van de Löcht, EMBO J. 9(1990), 2945-2950. Briefly, five day old subcultured parsley cells areused for the isolation of protoplasts. Protoplasting is achieved byovernight incubation of the cells in 0.24 M CaCl₂ containing 0.25% (w/v)cellulase and 0.05% (w/v) macerozyme at 24° C. Protoplasts are collectedby centrifugation (7 min., 100 g), washed with 0.24 M CaCl₂, and thenfloated in B5 medium (GIBCO/BRL) containing 0.4 M sucrose and 1 mg/ml2,4-dichlorophenoxyacetic acid. Protoplasts floating aftercentrifugation (5 min, 100 g) were harvested, counted and adjusted to2×10⁶/ml.

Supercoiled or linearized plasmid DNA (5-20 μg) containing the chimericpromoter-reporter (GUS) construct was transferred into the protoplastsusing the polyethylene glycol (PEG) method (Krens, Nature 296 (1982),72-74). Each transformation assay was split and placed into two 3 mlplates. The Pep25 (Nürnberger, Cell 78 (1994), 449-460) elicitor wasadded to one whereas the other served as a control. Both samples wereharvested after 8 hours, frozen in liquid nitrogen, crude proteinextracts prepared and GUS activity assayed (Jefferson, Plant Mol. Biol.Rep. 5 (1987), 387-405). Bradford assays (Bio-Rad) were used for proteindetermination. The expression data are given as mean fold inductionvalues±standard deviation (SD) and mean GUS activity (pmol/min/mg) fromsix independent transient transfection assays treated with or withoutPep25 elicitors.

5. Generation of Transgenic Plants

Transgenic plants were generated according to the methods described inBechtold, Mol. Biol. Genet. 316 (1993), 1194-1199; Grant, Science 269(1995), 843-846 and Dangl, Science 269 (1995), 843-846. Briefly, thepromoter elements were cloned in front of the reporter gene of thebinary vector pGPTV-GUS-kan (Becker, Plant Mol. Biol. 20 (1992),1195-1197) and the constructs introduced into the Agrobacterium strainGV3101 (pMP90; (Koncz and Schell, loc. cit.) containing the pMP90 helperplasmid. 500 ml cultures were grown in YEB medium containing kanamycin(50 μg/ml), rifampicin (100 μg/ml) and gentamycin (25 μg/ml). Cells wereresuspended in infiltration medium (0.5× Murashige-Skoog salts; 1×B5vitamins; 5.0% sucrose and 0.044 μM benzlaminopurine) and vacuuminfiltrated into Arabidopsis plants by the method of Grant (1995). T1seeds were surface-sterilized and transformants were selected on MSmedium containing 50 μg/ml kanamycin. Primary transformants weretransferred to soil and tested for GUS expression during pathogenesisand biotic or abiotic stress.

Example 1 Box S is Capable of Mediating Elicitor Induced Gene Expression

Box S (CAGCCACCAAAGAGGACCCAGAAT; SEQ ID NO: 7) has been shown to benecessary for the elicitor-responsive expression of the parsley eli 7genes (Takamiya-Wik, Ph.D. thesis, University of Köln, Köln, Germany(1995)). Together with the results concerning Box N (see Example 4.3)for the first time the core sequence of this type of element has beendefined which appears to be AGCCACCANA (SEQ ID NO: 14). The element isnot identical to any known elicitor-responsive element although it isvery similar to a number of ethylene response elements that have thecore sequence AGCCGCC (GCC Boxes) (Ohme-Takagi and Shinshi, The PlantCell 7 (1995), 173-182). In the promoters investigated (eli7-1, eli7-2and Prpl) there is always an A residue rather than a G. What differencethis difference in sequence makes is at present unclear and it is notknown whether Box S is responsive to ethylene. It has however been shownfor the first time that the Box S elements with the sequence AGCCACC areelicitor-responsive elements. The present data also show for the firsttime that GCC Boxes are also elicitor response elements as well as beingethylene response elements. Box S is a very strong elicitor-responsiveelement. A monomer of Box S gives 11-fold inducibility and a tetramer upto 560-fold inducibility. This clearly shows Box S to be an extremelypromising element for biotechnological purposes.

The sequence of the monomer element used is:5′-actagtCAGCCACCAAAGAGGACCCAGAATtctaga-3′ (SEQ ID NO: 19) with theelement in upper case letters and the SpeI/XbaI ends in lower caseletters. Constructs containing 1, 2, 4 and 8 copies of Box S wereconstructed and subjected to a transient expression assay as describedabove. The results were as follows:

Minus elicitor Plus elicitor Fold induction 1 × S 168 2058 12 2 × S 11810781 91 4 × S 187 76904 441 8 × S 781 102211 130

These Box S constructs are novel and have high inducibility. Four copiesof Box S appears to be the best with a very low background value (187) ahigh induced level (76904) and a very high fold induction (441×, thehighest of any of the constructs tested).

Example 2 Box D is Capable of Mediating Elicitor Inducible GeneExpression

Box D (TACAATTCAAACATTGTTCAAACAAGGAACC; SEQ ID NO: 11) is present in theparsley PR2 promoter and has never before been reported to be acis-acting element. Box D was identified by DNaseI footprinting, by lossof function experiments in the context of the PR2 promoter and bygain-of-function experiments with monomers and multimers. Box D is avery strong elicitor-responsive element, a tetramer directing 10-foldelicitor-inducibility combined with a very high level of expression,whilst a dimer is less strong but gives 15-20-fold inducibility. Thisclearly shows Box D to be a promising element for biotechnologicalpurposes.

The sequence of the element used is:5′-actagtTACAATTCAAACATTGTTCAAACAAGGAACCtctaga-3′ (SEQ ID NO: 20) withthe element in upper case letters and the SpeI/XbaI ends in lower caseletters. Constructs containing 1, 2 and 4 copies of Box D wereconstructed and subjected to the transient expression assay describedabove. The results were shown below.

Minus elicitor Plus elicitor Fold induction 1 × D 346 4002 11 2 × D 156231331 20 4 × D 5519 61552 11

These Box D constructs are novel. Two copies of Box D may be the bestwith a moderate background value (1562), a high induced level (31331)and a good fold induction (20×).

Example 3 Box U Provides for Elicitor Inducible Gene Expression

Box U (ATGAAGTTGAAATTCAATAG; SEQ ID NO: 13) is present in the parsleyPR2 promoter and has never before been reported to be a cis-actingelement. Box U has been defined by DNaseI footprinting, by loss offunction experiments in the context of the PR2 promoter and bygain-of-function experiments with monomers and multimers. Box U is areasonably strong elicitor-responsive element, a tetramer directing40-fold elicitor-inducibility.

The sequence of the element used is:5′-actagtAGTTGAAATTCAATAAGTTGAAATTCAATAtctaga-3′ (SEQ ID NO: 21) withthe element in upper case letters and the SpeI/XbaI ends in lower caseletters.

Constructs containing 2 copies of the above Box U sequence wereconstructed. The results of a transient expression assay are shownbelow. These therefore contain 4 copies of the Box U element(AGTTGAAATTCAATA; SEQ ID NO: 12). 1 or 2 copies of Box U are alsoactive.

Minus elicitor Plus elicitor Fold induction 4 × U 100 3947 39

These Box U constructs are novel. Box U appears to be a moderatelystrong pathogen-responsive element with a good fold induction (about40×).

Example 4 Some W Boxes are Capable of Mediating Elicitor Inducible GeneExpression

The results obtained in accordance with the present invention clearlyshow that there are great differences between the different W Boxes thathave been tested. Some are very strong (Box W2), some weak (Box W1),some are not active at all on their own (Box W3) and some are present ascomposite elements together with other cis-acting elements (Box N). TheW Boxes also have differences outside of the core TGAC sequences:

Box W1: (SEQ ID NO: 1) TTTGACC Box W2: (SEQ ID NO: 3) TTCAGCC-N₇-TTGACCBox W3: (SEQ ID NO: 5) TGAC-N₆-GTCA Box N: (SEQ ID NO: 8)TTTGACC plus GCCACC (S Box) Box W_(Amy): (SEQ ID NO: 6)TTGACC within TGAC-N₆-GTCA palindrome

4.1 Box W1

Box W1 (CACACTTAATTTGACCGAGTAACATTCGCC; SEQ ID NO: 2) has previouslybeen identified as a weak elicitor-responsive cis-element in the parsleyPR1 promoters and a tetramer has been shown to be sufficient to directelicitor-responsive expression in the parsley transient expressionsystem (Rushton, 1996). Box W1 contains the W box sequence TTGACC andevidence suggests that these elements are bound by the WRKY class oftranscription factors. As W boxes have also been found in the monocotsWild oat (Rushton, 1995) and maize (Raventos, 1995) and WRKY proteinshave been found in an increasing number of plant species this suggeststhat the W box elements may be cis-acting elements in all plant species.Box W1 had never before been tested on its own for activity as a monomeror in combination with other elements and it was observed that a monomerdirects elicitor-inducible expression (5-fold inducibility) and that BoxW1 is also active in combination with other elements (see below).

The current results show Box W1 itself, however, to be a weak element.The sequence of the element used (the monomer) is:5′-actagtCACACTTAATTTGACCGAGTAACATTCGCCtctaga-3′ (SEQ ID NO: 22) withthe element in upper case letters and the SpeI/XbaI ends in lower caseletters. This construct is slightly different than the constructpreviously reported (Rushton, 1996) as the element is inserted into theSpeI/XbaI sites and not BamHI/BglII. Constructs containing 1, 2 and 4copies of Box W1 were constructed and subjected to the transientexpression assay. The results were as follows.

Minus elicitor Plus elicitor Fold induction 1 × W1 362 1495 4.1 2 × W1299 2433 8.1 4 × W1 56 870 <15

The fold induction with 4×W1 is similar to the previously reportedvalues (Rushton, 1996). Comparison with values for other elements showsBox W1 to be a weak element.

4.2 Box W2

Box W2 (TTATTCAGCCATCAAAGTTGACCAATAAT; SEQ ID NO: 4) has previously beenidentified as a cis-acting element required for the elicitor responsiveexpression of parsley PR1 promoters in the transient expression system(Rushton, 1996). However, gain of function has been first demonstratedin accordance with the present invention. Box W2, like Box W1, containsa TTGACC element but the rest of the element is totally different andthese other sequences play an important role, as a tetramer of Box W1 isa weak element with about 10-fold elicitor inducibility whereas Box W2directs levels of expression up to 100 times higher than Box W1 with a50-fold elicitor inducibility. It is shown for the first time that BoxW2 alone, as a monomer or multimer, is a very strong elicitor-responsiveelement and that it is also active in combination with other elements.

The sequence of the element used (the monomer) is:5′-actagtTTATTCAGCCATCAAAGTTGACCAATAATtctaga-3′ (SEQ ID NO: 23) with theelement in upper case letters and the SpeI/XbaI ends in lower caseletters. Constructs containing 1, 2, 4 and 8 copies of Box W2 wereconstructed and subjected to the transient expression assay. Thefollowing results were obtained.

Minus elicitor Plus elicitor Fold induction 1 × W2 770 8914 11 2 × W2998 46651 46 4 × W2 2375 105685 44 8 × W2 7680 164454 21

W2 is the strongest elicitor-responsive cis-acting element that has beenso far tested, eight copies of W2 giving GUS values of approximately164,000.

4.3 Box N

Box N comes from the potato gst1 gene (TTCTAGCCACCAGATTTGACCAAAC; SEQ IDNO: 9) and has never previously been defined. It contains both an S Boxsequence (AGCCACCAGA) (SEQ ID NO: 24) and a W Box sequence (TTGACC)within just 25 base pairs and as such represents a novel cis-elementcomposed of two types of elicitor response element within a very smallstretch of DNA. A tetramer of Box N gives at least 75-fold elicitorinducibility. This observation suggests three important conclusions;firstly that Box N may be extremely useful for biotechnologicalapplications, secondly that the core Box S sequence is AGCCACCANA (SEQID NO: 14) and thirdly that Boxes S and W may represent a common themein plant promoters that respond to pathogens as these elements arepresent in both parsley and potato. Box N alone is a strongelicitor-responsive element and extremely interesting, as it consists ofan S Box (GCCACC) followed by a W Box (TTTGACC).

The sequence of the element used (the monomer) is:5′-actagtTTCTAGCCACCAGATTTGACCAAACtctaga-3′ (SEQ ID NO:18) with theelement in upper case letters and the SpeI/XbaI ends in lower caseletters. A construct with four copies of Box N was tested in transientexpression assay. The results were as follows.

Minus elicitor Plus elicitor Fold induction 4 × N 1085 92980 85

Box N is a strong element and shows a very high fold inducibility. Thisnovel combination and spacing of W and S Box elements may prove to bevery useful for biotechnological purposes.

4.4 Box W_(Amy)

Box W_(Amy) comes from the wild oat α-Amy2/A and wheat α-Amy2/54 geneswhere it has previously been published under the name Box 2 or O2S (seeRushton, Plant Mol. Biol. 29 (1995), 691-702). It is a cis-actingelement required for the transcriptional activation of these genesduring germination but has never previously been linked to a role inpathogenesis. Box W_(Amy) consists of two W Box elements: a hexamer5′-TTGACC-3′ embedded in a palindromic 5′-TGAC-N₆-GTCA-3′. As itcontains both types of sequences together it constitutes a new type of WBox and may be a “super W Box.”

The sequence of the element used (the monomer) is:5′-actagtGGATTGACTTGACCGTCATCGGCTtctaga-3′ (SEQ ID NO: 25) with theelement in upper case letters and the SpeI/XbaI ends in lower caseletters. A construct containing 7 copies of Box W_(Amy) was constructedand to the transient expression assay. The result is shown below.

Minus elicitor Plus elicitor Fold induction 7 × W_(Amy) 168 43867 260

W_(Amy) is a strong elicitor-responsive cis-acting element and has thehighest fold induction of any W Box that has been so far tested. Thiselement could therefore be a particularly effective W Box and could aidthe designing of synthetic W Boxes that are even more effective.

Example 5 Synthetic Promoters Consisting of Combinations of theAbove-Described Elements

Synthetic promoters composed of combinations of the aboveelicitor-responsive elements have never before been constructed ortested. All elements (Boxes W1, W2, S, U, D, N and W_(Amy)) are activein combination with each other; monomer, dimer and tetramer constructsbeing active. The furthest downstream element (nearest to the TATA Box)has the strongest effect on the synthetic promoter with further upstreamelements having a much lesser effect. However the combination of two ormore different types of cis-element may have a much more profound effecton expression in planta. In addition the insertion of a spacer regioncomposed of anything between 100 base pairs and 1,000 base pairs appearsto increase the contribution of the more upstream cis-elements. All ofthese synthetic promoters are good candidate promoters that may berapidly and locally responsive to pathogen attack but also shownegligible activity in uninfected tissues. These promoters may thereforeallow the engineering of defense reactions that are closely related tonatural defense mechanisms without appreciable activity in non-infectedcells of the plant.

A large number of combinations have been tested. The results for some ofthese are detailed below. All of these combinations are novel and theseconstructs represent true synthetic promoters. The elements are insertedinto the SpeI/XbaI sites, as with all of the constructs, and read fromthe 5′ end to the 3′ end i.e. 4×W2/4×S is:

SpeI-W2-W2-W2-W2-S-S-S-S-XbaI

Generally, the elements nearest to the TATA Box (i.e. at the 3′ end)have the greatest effect on both level of expression and fold induction.The effect of the upstream elements is often minimal and there is alsoan inhibitory effect probably due to steric hindrance when differentelements are put close together; compare 4×S/4×W2 with (2×S/2×W2)×2. Theinsertion of spacer regions between elements is therefore recommended toalleviate problems due to steric hindrance. The results of the transientexpression assays are shown below.

Minus elicitor Plus elicitor Fold induction 1 × S/1 × W2 1732 85126 49 2× S/2 × W2 1529 95872 62 4 × S/4 × W2 2654 64105 24 (2 × S/2 × W2) × 2483 9832 20 4 × W2/4 × S 2753 205826 74 1 × W2/1 × S 146 2690 18 2 × S/2× D 191 15541 81 4 × S/4 × D 9775 100265 10 1 × D/1 × S 32 1246 38 4 ×D/4 × S 6795 204115 30 2 × W2/2 × D 1762 32462 18 4 × W2/4 × D 2204292875 4.2 4 × D/4 × W2 18857 276456 14 1 × D/1 × W2 295 4369 14

Adding more copies of an element in a composite construct oftenincreases the absolute level of expression (e.g. 2×W2/2×D and 4×W2/4×D)but often lowers the fold induction. In some cases even the absolutelevel of expression decreases (e.g. 2×S/2×W2 and 4×S/4×W2) and acomparison with (2×S/2×W2)×2 suggests that this is due to sterichindrance as the number of copies of the elements is the same, it isjust the order that is changed.

Example 6 Box E17 is Capable of Mediating Elicitor Induced GeneExpression

Box E17 (TCAATATGTCAATGGTCAACATTCAAC; SEQ ID NO: 15) was isolated fromthe promoter of the parsley Eli17 gene which is known to react toelicitor-treatment with transcript accumulation (Somssich, Plant Mol.Biol. 12 (1989), 227-234). Recently it has been shown that the Eli17gene reacts very rapidly and transiently to elicitor-treatment andpathogen infection. This has never been previously described.

The sequence of the monomer element used is:5′-actagtTCAATATGTCAATGGTCAACATTCAACtctaga-3′ (SEQ ID NO: 26) with theelement in upper case letters and the SpeI/XbaI ends in lower caseletters. Constructs containing 1 and 2 copies of Box E17 as well as amonomeric reverse complement of Box E17 were constructed (FIG. 4,constructs B109, A109, and 18S102, respectively) and subjected to atransient expression assay as described above. As shown in FIG. 4, themonomer has 5-fold inducibility and the dimer 50-fold. In comparison tothe other cis-elements of the present invention moderate induction wasachieved by Box E17. Likewise, a tetramer of Box E17 was subjected totransient assays (data not shown), which resulted in 5- to 20-foldinduction following elicitor-treatment. However, this result cannot becompared to the induction values of the Box E17 constructs mentionedabove because of diminished quality of the parsley protoplasts used.Presumably, the Box E17 tetramer mediates at least an induction ashigh-fold as the respective dimer.

Similar to cis-elements of Example 4, Box E17 contains two copies of theW-Box core motif TGAC, in reverse orientation (GTCA) as tandem repeatseparated by a 3 bp spacer. The importance of this core motif can beinferred from preliminary mutagenesis experiments (FIG. 4, constructsC109, 17S102, and 15S102). A 1 bp deletion within the W-Box motifresulted in complete loss of function in contrast to deletions at twodifferent sites having no effect to inducibility. In order to furthernarrow down the minimal structure capable of mediatingelicitor-responsiveness dissected Box E17 elements were tested intransient expression assays as described above. The initial Box E17 (SEQID NO: 15) was deleted from the 5′-end by 6 bp, from the 3′-end by 7 bpand from both ends by 6 and 7 bp, respectively. Each of theseoligonucleotides were ligated into the BamHI site of the MS23 vector,which was before cut with BamHI restriction enzyme and the overhangsblunted, giving rise to the promoter constructs H149, B175, C175 andD175 (FIG. 5). The promoter constructs showed remarkable differencesregarding their elicitor-responsiveness. C175 and D175 having the 3′-endtruncated, displayed no significant induction upon elicitor treatment.On the other hand, the 5′-truncated B175 gave values which were similarto those of the 27 bp Box E17 element. Thus, also the 21 bp-element B175(SEQ ID NO: 16) is a functional cis-element in the sense of the presentinvention.

Furthermore, Box E17 is not only sufficient but also necessary to lendthe Eli17 promoter, or at least a 445 bp long functional part thereofwhich comprises said element naturally, its elicitor inducibility. Thiscould be shown in transient expression assays which were performed withan MS23-construct containing the 445 bp stretch having the 27 bp-elementremoved. The resulting complete loss of elicitor-dependent inducibility(see FIG. 5) indicates the crucial role of Box E17 for elicitor- andpathogenesis-related gene regulation in its natural environment andfurther supports its applicability for conferring inducibility uponelicitation or pathogenesis to a chimeric promoter according to thepresent invention.

Example 7 Chimeric Promoters with Varying Distances of the Box E17Element to the Minimal Promoter are Inducible

In order to elucidate the optimal position of the Box E17 element withinthe chimeric promoter several constructs with varying distances to the35S minimal promoter were tested (FIGS. 6, 7 a and 7 b). For thispurpose Box E17 was inserted into different restriction sites of thems23 polylinker. After digesting the vector and filling in theoverhangs, the cis-element was blunt ligated into the respective site asa monomer or as a dimer. The transient assays were conducted asdescribed above. The results (FIGS. 7a and 7b ) indicate an optimaldistance of Box E17 to the 5′ end of the minimal promoter of 40 to 60 bp(corresponding to the restriction sites BamHI, ClaI, EcoRI). Still goodinduction was observed for the SapI site in 131 bp distance whereasconsiderably weaker response was obtained when Box E17 was inserted intothe SalI site which is 5 bp upstream of the minimal promoter.

Example 8 Transgenic Plants Carrying Chimeric Promoters

Transformants were tested for the response of the synthetic promoters topathogens. Cultures of the bacterium pseudomonas (strains Rpt2 or Rpm1)were grown in King's-B Medium containing 30 μg/ml kanamycin and 100μg/ml rifampicin. The bacteria were resuspended in 10 mM MgCl₂ at anOD₆₀₀ of 0.2 and infiltrated into leaves via a syringe. Controls wereperformed using 10 mM MgCl₂ alone. After 6 hours the leaves were removedfrom the plants and stained for GUS activity using X-Gluc. Theexpression pattern observed in the transgenic plants containing the GUSmarker gene under the control of the chimeric promoter of the inventionrevealed expression in tissue infected by Pseudomonas syringae and insome cases also local expression in wounded tissues.

With regard to Box E17 a chimeric promoter comprising the dimer of thiselement (A109, FIG. 4) and the 35S minimal promoter was used fortransformation of Arabidopsis plants. Two to three weeks old seedlingsand old leaves of the transformants were infiltrated with a 10 μMaqueous solution of the bacterial elicitor Flagellin 22 via a syringe(Felix, Plant Journal 18 (1999) 262-276; Gömez-Gömez, Plant Journal 18(1999) 277-284) which led to clear GUS activation. High induction wasalso observed after infection by a fungal (Peronospora parasitica) and abacterial pathogen (Pseudomonas syringae).

Peronospora infections were carried out according to Dangl et al.(Genetic definition of loci involved in Arabidopsis-pathogeninteractions. In: Methods in Arabidopsis Research (Koncz, Chua andSchell, eds.). Singapore: World Scientific Publishing Co. (1992),393-418) or Koch (Plant Cell 2 (1990), 437-446).

On the other hand, mechanical stress induced for example by wounding didnot activate the chimeric promoter. And surprisingly, no or only mereexpression and activation of the reporter gene was observed in rootwhich is the organ where the Eli17 gene is predominantly expressed inparsley. Thus, organ specificity appears not to be mediated by Box E17.

Furthermore, expression studies were performed the results of which aresummarized in FIG. 8. Seven different tetramers of cis-elements wereassayed for their background expression in aerial parts and roots,respectively, and for their inducibility after wounding, senescence,incompatible and compatible Peronospora infection. Some importantconclusions can be drawn from these experiments:

All of these chimeric promoters that are inducible by incompatiblestrains of Peronospora parasitica are also inducible by compatiblestrains. This is an important observation regarding the presentinvention as it shows that these constructs could be inducible by allpotential pathogens and not just those for which there is already afunctional defense system in operation in the plant.

Although many constructs show induced expression around infection sites,the expression characteristics are different with, for example, some WBoxes (e.g. W2) being expressed in an area around the infection sitewhereas others are expressed within the infection site itself. This isan unexpected finding as it shows that within a class of cis-actingelements (W Boxes or GCC/S Boxes) differences in sequence outside of thecore sequence lead to differences in functionality.

All of the cis-acting elements of the present invention show inducibleexpression in a heterologous plant (Arabidopsis). As these elements comefrom parsley, potato and wheat this clearly shows that these elementscould be functional in all plants. This general functionality of suchelements is an important new observation.

The invention also pertains to the following exemplary embodiments:

1. A chimeric promoter capable of mediating local gene expression inplants upon pathogen infection comprising (i) at least one cis-actingelement sufficient to direct elicitor-specific expression comprising thenucleotide sequence of any one of SEQ ID NOS: 3 to 16, and (ii) aminimal promoter.

2. The chimeric promoter of embodiment 1, further comprising acis-acting element having the nucleotide sequence of SEQ ID NO: 1 or 2.

3. The chimeric promoter of embodiment 1 or 2, wherein said syntheticplant promoter comprises homo- and/or hetero-multimeric forms of saidcis-acting element (s).

4. The chimeric promoter of any one of embodiments 1 to 3, wherein saidmultimeric form is a dimer or tetramer.

5. The chimeric promoter of any one of embodiments 1 to 4, wherein theminimal promoter is derived from the CaMV35S promoter, CHS promoter, PR1promoter, or hcbt2 promoter.

6. The chimeric promoter of any one of embodiments 1 to 5, wherein thedistance between said cis-acting element and said minimal promoter is 12to 300 base pairs, more preferably 25 to 70 base pairs, and mostpreferably 40 to 60 base pairs.

7. The chimeric promoter of any one of embodiments 1 to 6, wherein aspacer region composed of 4 to 10 base pairs separates at least two ofsaid cis-acting elements.

8. The chimeric promoter of any one of embodiments 3 to 7, wherein atleast two of said multimeric forms are separated by a spacer of betweenabout 50 to 1000 base pairs.

9. The chimeric promoter of any one of embodiments 1 to 8, wherein theinduction of gene expression upon elicitor treatment or pathogeninfection is at least 15-fold.

10. A recombinant gene comprising the chimeric promoter of any one ofembodiments 1 to 9.

11. The recombinant gene of embodiment 10, wherein the chimeric promoteris operatively linked to a heterologous DNA sequence.

12. The recombinant gene of embodiment 10 or 11, wherein at least one ofsaid cis-acting elements is located in the 5′- or 3-untranslated regionor in an intron of the recombinant gene.

13. The recombinant gene of embodiment 11 or 12, wherein saidheterologous DNA sequence encodes a (poly) peptide, cytotoxic protein,antibody, antisense RNA, sense RNA, ribozyme, transcription factor,protease, nuclease, lipase, or polymerase.

14. A vector comprising the chimeric promoter of any one of embodiments1 to 9 or the recombinant gene of any one of embodiments 10 to 13.

15. A method for the production of transgenic plants, plant cells orplant tissue comprising the introduction of a chimeric promoter of anyone of embodiments 1 to 9, a recombinant gene of any one of embodiments10 to 13 or the vector of embodiment 14 into the genome of said plant,plant cell or plant tissue.

16. Plant cells comprising a chimeric promoter of any one of embodiments1 to 9, the recombinant gene of any one of embodiments 10 to 13 or thevector of embodiment 14 or obtainable by the method of embodiment 15.

17. A transgenic plant or plant tissue comprising plant cells ofembodiment 16.

18. The transgenic plant of embodiment 17, which upon the presence ofthe chimeric promoter or the recombinant gene attained resistance orimproved resistance against a pathogen the corresponding wild-type plantwas susceptible to.

19. Harvestable parts of a transgenic plant of embodiment 17 or 18comprising plant cells of embodiment 16.

20. Propagation material of a transgenic plant of embodiment 17 or 18comprising plant cells of embodiment 16.

21. A cis-acting element as defined in embodiment 1 or a multimeric form(s) of any one of those as defined in embodiment 3 or 4.

22. A method for the identification of an activator or inhibitor ofgenes specifically expressed in plants upon pathogen infectioncomprising the steps of: (a) providing a plant, plant cell, or planttissue comprising a recombinant DNA molecule comprising a readout systemoperatively linked to the chimeric promoter of any one of embodiments 1to 9; (b) culturing said plant cell or tissue or maintaining said plantin the presence of a compound or a sample comprising a plurality ofcompounds under conditions which permit expression of said readoutsystem; (c) identifying or verifying a sample and compound,respectively, which leads to suppression or activation and/orenhancement of expression of said readout system in said plant, plantcell, or plant tissue.

23. The method of embodiment 22 further comprising the step of (d)subdividing the samples identified in step (c) and repeating steps (a)to (c) one or more times.

24. The method of embodiment 22 or 23 further comprising the step of (e)identifying and/or isolating from the identified sample the compoundresponsible for said suppression or activation and/or enhancement ofexpression of said readout system in said plant, plant cell, or planttissue.

25. The method of any one of embodiments 22 to 24, wherein (a) saidrecombinant DNA molecule is a recombinant gene of any one of embodiments10 to 13 or a vector of embodiment 14; (b) said plant cell is a plantcell of embodiment 16; (c) said plant tissue is a plant tissue ofembodiment 17, or (d) said plant is a plant of embodiment 17 or 18.

26. A method for preparing a plant elicitor comprising the steps of themethod of any one of embodiments 22 to 25 and formulating the compoundobtained or identified in step (c) or (e) in a form suitable for theapplication in agriculture or plant cell and tissue culture.

27. A compound obtained or identified by the method of any one ofembodiments 22 to 26 which is an activator or inhibitor of geneexpression and/or function in plants.

28. An antibody specifically recognizing the compound of embodiment 27or the cis-acting element of embodiment 21.

29. A diagnostic composition comprising a chimeric promoter of any oneof embodiments 1 to 9, the recombinant gene of any one of embodiments 10to 13, the vector of embodiment 14, the compound of embodiment 27 or theantibody of embodiment 28, and optionally suitable means for detection.

30. A kit comprising a chimeric promoter of any one of embodiments 1 to9, the recombinant gene of any one of embodiments 10 to 13, the vectorof embodiment 14, the compound of embodiment 27 or the antibody ofembodiment 28.

31. A plant protection composition comprising the compound of embodiment27.

32. Use of a cis-acting element sufficient to direct elicitor-specificexpression, a chimeric promoter of any one of embodiments 1 to 9, therecombinant gene of any one of embodiments 10 to 13, the vector ofembodiment 14, the cis-acting element of embodiment 21 and/or thecompound of embodiment 27 for the production of pathogen resistantplants.

33. Use of a cis-acting element sufficient to direct elicitor-specificexpression, the chimeric promoter of any one of embodiments 1 to 9, arecombinant gene of any one of embodiments 10 to 13, a vector ofembodiment 14, the plant cell of embodiment 16, the plant tissue ofembodiment 17, or the plant of embodiment 17 or 18 for identifyingand/or producing compounds capable of conferring induced resistance to apathogen in a plant.

34. A method of rendering a gene responsive to pathogens comprisinginserting at least one cis-acting element sufficient to directelicitor-specific expression into the promoter of said gene.

35. A method for preparing a promoter capable of mediating local geneexpression in plants upon pathogen infection comprising operably linkinga cis-acting element sufficient to direct elicitor-specific expressionto a transcription initiation sequence of a promoter.

36. The method of embodiment 34 or 35, wherein said cis-acting elementis a cis-acting element as defined in embodiment 1 or 2 or a multimericform thereof as defined in any one of embodiments 3 to 8.

37. The method of any one of embodiments 34 to 36, further comprisingdeleting non-specific cis-acting elements in the promoter.

38. The promoter obtainable by the method of any one of embodiments 34to 37.

39. Use of the compound of embodiment 27 as plant protective agent orherbicide.

It will be appreciated by persons skilled in the art that the presentinvention is not limited to what has been particularly shown anddescribed herein above. In addition, unless mention was made above tothe contrary, it should be noted that all of the accompanying drawingsare not to scale. A variety of modifications and variations are possiblein light of the above teachings without departing from the scope andspirit of the invention, which is limited only by the following claims.

What is claimed is:
 1. An isolated cis-acting element sufficient todirect pathogen-elicitor-specific expression,pathogen-infection-specific expression, or both, consisting of thenucleotide sequence of SEQ ID NO: 8 or
 9. 2. A chimeric promoter capableof local gene expression in plants of an operably linked nucleic acidsequence, wherein the expression is induced by a pathogen elicitortreatment, a pathogen infection, or both, wherein the chimeric promotercomprises: (i) at least two cis-acting elements sufficient to direct:the pathogen-elicitor-specific expression of the nucleic acid sequence,the pathogen-infection-specific expression of the nucleic acid sequence,or both, wherein two of the at least two cis-acting elements are each acis-acting element consisting of the nucleotide sequence of SEQ ID NO: 8or 9, and (ii) a minimal promoter, wherein each of said two of the atleast two cis-acting elements consisting of the nucleotide sequence ofSEQ ID NO: 8 or 9 are separated by only a spacer of from 4 to 10 basepairs.
 3. The chimeric promoter according to claim 2, further comprisingat least one additional cis-acting element, wherein said at least oneadditional cis-acting element consists of the nucleotide sequenceselected from the group consisting of the nucleotide sequences of one ofSEQ ID NO: 1-16.
 4. The chimeric promoter of claim 3, wherein saidchimeric promoter comprises at least one of homo- and hetero-multimericforms of at least one of the at least two cis-acting elements and saidat least one additional cis-acting element.
 5. The chimeric promoter ofclaim 4, wherein said homo- and/or hetero-multimeric form is a dimer ora tetramer.
 6. The chimeric promoter of claim 3, wherein at least two ofthe elements, selected from said at least two cis-acting elements andsaid at least one additional cis-acting element, are separated by aspacer of about 50 to about 1000 base pairs.
 7. The chimeric promoteraccording to claim 2, wherein at least one of the at least twocis-acting elements comprises two copies of the nucleotide sequence ofSEQ ID NO: 8 or
 9. 8. The chimeric promoter according to claim 7,further comprising two copies of the nucleotide sequence of SEQ ID NO: 3or
 4. 9. The chimeric promoter of claim 2, wherein the minimal promoteris a minimal promoter from a promoter selected from the group consistingof a CaMV35S promoter, a CHS promoter, a PR1 promoter, and a hcbt2promoter.
 10. The chimeric promoter of claim 2, wherein the distancebetween at least one of said at least two cis-acting elements and saidminimal promoter is 12 to 300 base pairs.
 11. The chimeric promoter ofclaim 2, wherein the distance between said at least two cis-actingelements and said minimal promoter is 25 to 70 base pairs.
 12. Thechimeric promoter of claim 2, wherein the distance between said at leastone cis-acting element and said minimal promoter is 40 to 60 base pairs.13. A recombinant gene comprising the chimeric promoter of claim
 2. 14.The recombinant gene of claim 13, wherein at least one of the at leasttwo cis-acting elements is located in the 5′- or 3′-untranslated regionor in an intron of the recombinant gene.
 15. The recombinant gene ofclaim 13, wherein the chimeric promoter is operatively linked to aheterologous DNA sequence, and wherein said heterologous DNA sequenceencodes a (poly)peptide, a cytotoxic protein, an antibody, an antisenseRNA, a sense RNA, a ribozyme, a transcription factor, a protease, anuclease, a lipase, or a polymerase.
 16. A vector comprising thechimeric promoter of claim 2 or the recombinant gene of claim
 13. 17. Atransgenic plant cell comprising the chimeric promoter, a recombinantgene comprising the chimeric promoter, or a vector comprising thechimeric promoter of claim
 2. 18. A transgenic plant or a plant tissuecomprising the transgenic plant cell of claim
 17. 19. A harvestable partof a transgenic plant of comprising the transgenic plant cell of claim17.
 20. A propagation material of a transgenic plant of comprising thetransgenic plant cell of claim 17.